Wearable C peptide detection device and detection method and application thereof

Abstract

The invention discloses a wearable C peptide detection device as well as a detection method and application thereof. The device comprises a detection chip, and the detection chip comprises a microneedle array, a reaction cavity and an injection part which are sequentially connected through a liquid outlet channel. According to the wearable C peptide detection device, rapid detection of C peptide can be achieved within 15 min, and meanwhile, the wearable C peptide detection device can be repeatedly used after elution, so that repeated, rapid and dense data collection can be achieved, and real-time monitoring of the C peptide is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of intelligent detection equipment, and in particular to a wearable C-peptide detection device and a detection method and application thereof. Background Art

[0002] C-peptide is a straight chain composed of 31 amino acids with a molecular weight of 3020. C-peptide, also known as connecting peptide, is secreted by pancreatic β cells. It has a common precursor with insulin - proinsulin. One molecule of proinsulin will be broken down into one molecule of insulin and one molecule of C-peptide, so the molar amount of C-peptide is consistent with that of one's own insulin. However, since C-peptide is not easily degraded by the liver, its half-life is 3-4 times that of insulin. And because of its longer half-life and consistent source, the content of self-produced insulin can be detected by detecting the content of C-peptide, which can accurately reflect the secretory function of the body's pancreatic β cells. For patients receiving insulin treatment, measuring the level of insulin in the blood cannot evaluate their own pancreatic function, so measuring the level of C-peptide is a very important method for evaluating their own pancreatic β cell function.

[0003] At present, the detection of C-peptide is not perfect in the classification and early diagnosis of diabetes, the establishment and adjustment of treatment plans, and the study of the pathogenesis of diabetes. At present, the main C-peptide detection technologies include: enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, liquid chromatography-mass spectrometry (LC-MS / MS), radioimmunoassay (RIA) and turbidimetric immunoassay. However, these methods have great defects. For example, the detection time of ELISA is long, usually more than 1 hour, and it requires relatively expensive readout equipment and professional operation. In addition, the standardization of its determination may vary between different laboratories, affecting the comparability of the results. Although chemiluminescent immunoassay has high sensitivity, the equipment cost is high and the skill requirements of the operator are high. In addition, the stability and storage of chemiluminescent reagents may also affect the accuracy of the test results. Liquid chromatography-mass spectrometry provides very high accuracy and specificity, but its equipment is expensive, the operation is complicated, and it requires professional operators, and it is not suitable for routine clinical testing. Radioimmunoassay (RIA) involves radioactive substances and poses potential health risks to the environment and operators. In addition, the stability of radioactive labels and waste disposal are also important considerations in the use of RIA. Turbidimetric immunoassays have low sensitivity and accuracy and are not suitable for clinical decisions that require high precision. Its application is more limited to resource-constrained environments. In summary, the current clinical methods for detecting C-peptide are usually limited by the need for professional operators and expensive instruments and equipment. In addition, these test results only intelligently reflect the C-peptide level at a specific time point (when blood or urine is drawn), and cannot be monitored continuously in real time. They are severely restricted by time and space and lack portability.

[0004] Therefore, it is of great significance to develop a method that can solve the pain points of the above-mentioned clinical C-peptide detection methods and propose a portable C-peptide monitoring device that does not require professional personnel, is inexpensive, is not restricted by time and space, and can continuously monitor changes in one's own C-peptide. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide a wearable C-peptide detection device and its detection method and application. The wearable C-peptide detection device in the present invention can realize rapid detection of C-peptide within 15 minutes, and at the same time, it can be used repeatedly after elution, so as to realize repeated, rapid and intensive data collection and realize real-time monitoring of C-peptide.

[0006] A first aspect of the present invention provides a detection chip, which includes a microneedle array, a reaction chamber, and an injection portion that are sequentially connected through a liquid outlet channel.

[0007] In some embodiments of the present invention, the microneedle array is used to obtain a test sample from the skin.

[0008] In some embodiments of the present invention, the reaction chamber is pre-buried with fixed capture objects.

[0009] In the present invention, the term "immobilization" means that a substance is fixed or linked to a target by methods known in the art, and at least during the reaction process or under the reaction conditions, it will not be eluted or inactivated by any substance involved in the reaction.

[0010] In some embodiments of the present invention, the immobilization is performed using biotin-avidin. In some embodiments of the present invention, the immobilization of the captured object can be achieved by modifying the surface of the reaction chamber with biotin or avidin and modifying the captured object with another corresponding substance.

[0011] In some embodiments of the present invention, the capture object is used to capture a substance to be detected in a test sample.

[0012] In some embodiments of the present invention, the injection part is loaded with a reaction liquid for storing and driving the stored reaction liquid to react with the substance to be detected captured by the capture object in the reaction chamber.

[0013] In some embodiments of the present invention, the reaction solution includes a conjugate, a color developer, a washing solution and an eluent.

[0014] In some embodiments of the present invention, the conjugate is used to bind to a substance to be detected or a substance to be detected-capture complex.

[0015] In some embodiments of the present invention, the eluent is used to dissociate the substance to be detected and the captured substance.

[0016] In some embodiments of the present invention, the eluent is glycine or its hydrochloride.

[0017] In the present invention, the eluting agent can achieve complete elution of the substance to be detected and the conjugate, thereby re-exposing the capture object and using it for new detection.

[0018] In some embodiments of the present invention, the color developing solution is TMB color developing solution. Of course, those skilled in the art can select other conventional color developing solutions in the art according to the use requirements.

[0019] In some embodiments of the present invention, the washing solution includes phosphate buffered saline (PBS), Tris buffered saline (TBS), or other conventional commercially available antibody diluents in the art.

[0020] In some embodiments of the present invention, the microneedles in the microneedle array are porous microneedles with 2 to 3 holes.

[0021] In some embodiments of the present invention, the number of holes is 2.

[0022] In some embodiments of the present invention, the diameter of the pores is 120-180 μm.

[0023] In some embodiments of the present invention, the diameter of the pore is 140-160 μm.

[0024] In some embodiments of the present invention, the diameter of the pore is 150 μm.

[0025] In some embodiments of the present invention, the microneedle spacing is 1100-1300 μm.

[0026] In some embodiments of the present invention, the microneedle spacing is 1200 μm.

[0027] In some embodiments of the present invention, the bottom diameter of the microneedle is 500-700 μm, and the vertical height is 500-700 μm.

[0028] In some embodiments of the present invention, the bottom diameter of the microneedle is 600 μm and the vertical height is 650 μm.

[0029] In some embodiments of the present invention, the capture object and the binder include any substance that can specifically bind to the substance to be detected, including: antigen-antibody, enzyme-substrate, complementary pairing sequence and biotin-avidin.

[0030] In some embodiments of the present invention, the capture object and the binding object are antigen-antibody.

[0031] In some embodiments of the present invention, the capture object and the binding object are antibody A and antibody B of the substance to be detected.

[0032] In some embodiments of the present invention, the antibody A and the antibody B are a pair of antibodies against a substance to be detected.

[0033] In some embodiments of the present invention, the substances to be detected include proteins, nucleic acid molecules and drug molecules.

[0034] In some embodiments of the present invention, the substance to be detected is C-peptide.

[0035] In the present invention, taking C-peptide as an example, a wearable portable detection device is constructed. Those skilled in the art can select appropriate capture substances and binders for replacement based on the category of the object to be detected to obtain a detection device for a specific object to be detected.

[0036] In some embodiments of the present invention, the detection chip further includes a suction device and a pressure release channel.

[0037] In some embodiments of the present invention, the suction device is used to assist the microneedle array in obtaining the test sample.

[0038] In some embodiments of the present invention, the suction device is a syringe. Of course, those skilled in the art can use other suction devices to replace it according to the use requirements to achieve the same or similar effects.

[0039] In some embodiments of the present invention, the pressure release channel is used to release pressure on the detection chip.

[0040] In some embodiments of the present invention, a controllable switch is provided on the pressure release channel, and the controllable switch includes a valve device. Of course, those skilled in the art can also use other controllable switches to open and close the channel, including manual, semi-automatic or fully automatic switches.

[0041] In some embodiments of the present invention, a controllable switch is further provided on the liquid outlet channel for controlling the flow of the fluid in the injection portion to the reaction chamber. In some embodiments of the present invention, the controllable switch comprises a valve device. Of course, those skilled in the art may also use other controllable switches to realize the opening and closing of the channel, including manual, semi-automatic or fully automatic switches.

[0042] In some embodiments of the present invention, all channel sizes are not limited and can be adjusted according to usage requirements.

[0043] In some embodiments of the present invention, the injection part contains a plurality of injection cavities. In some embodiments of the present invention, the volume and size of the injection cavity are not limited and can be adjusted according to the use requirements.

[0044] In some embodiments of the invention, the injection chamber comprises a syringe.

[0045] In some embodiments of the present invention, the reaction solutions are loaded into different injection chambers respectively.

[0046] In some embodiments of the present invention, the detection chip is specifically composed of a microneedle array, a reaction chamber and a plurality of syringes, wherein at least one syringe is used as a suction device, and the remaining syringes are used as injection parts to load the reaction solution. The reaction solutions are loaded into different syringes according to their types. The syringes loaded with the reaction solutions are connected in parallel through the liquid outlet channels, and the flow of the fluid to the reaction chamber is controlled by valves. The suction device and the microneedle array are each independently connected to the reaction chamber through a channel (i.e., not in parallel with other channels). At the same time, the reaction chamber is also independently provided with a pressure release channel, which is controlled to be opened and closed by a valve.

[0047] In some embodiments of the present invention, the detection chip can be prepared by conventional methods in the art, including but not limited to 3D printing and casting into a mold.

[0048] In some embodiments of the present invention, the structural schematic diagram of the detection chip is as follows Figure 1 shown.

[0049] A second aspect of the present invention provides a detection device, which includes the above-mentioned detection chip and a fixing device, wherein the fixing device is used to fix the detection chip on the surface of the part to be sampled.

[0050] In some embodiments of the present invention, the fixing device may be independent of the detection chip, or assembled with the detection chip.

[0051] The third aspect of the present invention provides the use of the above-mentioned detection chip or the above-mentioned detection device in real-time detection or monitoring in vitro.

[0052] In some embodiments of the present invention, the in vitro real-time detection or monitoring does not involve the diagnosis of a disease.

[0053] In some embodiments of the present invention, the objects of in vitro real-time detection or monitoring include but are not limited to proteins, nucleic acid molecules and drug molecules.

[0054] In some embodiments of the present invention, the object of in vitro real-time detection or monitoring is C-peptide.

[0055] In the present invention, the real-time detection or monitoring data of C-peptide can be further used as a reference for specific aspects, including but not limited to:

[0056] 1. Evaluation of the secretory function of pancreatic β cells: Since there is a fairly stable proportional relationship between C-peptide and insulin, and it is not interfered by insulin antibodies, and the injected exogenous insulin does not contain C-peptide, accurate measurement of C-peptide levels can analyze the level of insulin produced by the body itself, thereby further understanding the function of β cells. Since insulin and C-peptide are secreted by pancreatic β cells at equimolar molecular concentrations, and C-peptide is rarely taken up by the liver, is slowly degraded in peripheral blood, and C-peptide does not cross-react with insulin antibodies, the C-peptide value can more accurately reflect the reserve and secretory function of pancreatic β cells.

[0057] 2. Assist in determining the type of diabetes: The C-peptide levels of type 1 diabetes are low at all times, both fasting and after meals, because the secretion function of pancreatic β cells in patients with type 1 diabetes is seriously defective. However, the fasting C-peptide value of type 2 diabetes patients is not low, and may even be higher than that of normal people. However, the C-peptide value is lower than normal after sugar load, and the peak value is delayed to 2 hours later. The reason is that their insulin release function is poor and the pancreatic β cell reserve function is insufficient. Through the relationship between C-peptide, insulin, and pancreatic β cells, it can be used to assist in the classification of diabetes, thereby guiding clinical medication, such as insulin therapy or other oral drugs.

[0058] 3. Auxiliary identification of hypoglycemia: As above, through the relationship between C-peptide, insulin and pancreatic beta cells, it can be analyzed and found that if the C-peptide value exceeds normal, it can be speculated that the hypoglycemia is caused by excessive insulin secretion; if the C-peptide value is lower than normal, it is caused by other reasons and other tests can be further performed.

[0059] 4. Assist in the diagnosis of islet cell tumors and determine the effect of insulinoma surgery: As above, through the relationship between C-peptide, insulin, and pancreatic β cells, it can be analyzed and found that the C-peptide level in the blood of insulinoma is high, generally >4.0ng / mL. If the C-peptide level in the blood is still high after surgery, it means that there is residual tumor tissue. If the C-peptide level continues to rise during follow-up, it indicates that the tumor may recur or metastasize, which can assist in reminding physicians to conduct inspections and diagnoses in this regard.

[0060] 5. Islet transplantation: Patients who receive islet transplantation generally need to use insulin treatment. Therefore, through the relationship between C-peptide, insulin, and pancreatic β cells, it can be analyzed and found that the measurement of C-peptide can understand the secretory function of β cells after transplantation.

[0061] 6. Assist in the assessment of liver and kidney diseases: When suffering from hepatitis or cirrhosis, the liver's insulin uptake is reduced, and the blood insulin level tends to increase, while C-peptide is less affected, and the ratio of C-peptide to insulin in the blood decreases. Therefore, through the relationship between C-peptide, insulin, and pancreatic β cells, it can be analyzed and found that when kidney disease occurs, C-peptide degradation slows down, blood C-peptide levels increase, and the ratio of C-peptide to insulin is significantly higher than normal, which can assist doctors in the examination and diagnosis of this aspect.

[0062] A fourth aspect of the present invention provides a cyclic detection method, comprising:

[0063] Add the test sample and the binder to the pre-embedded capture object. After a period of reaction (10 minutes), wash away the unbound test sample and the binder. Add TMB for color development for 5 minutes and read the test result. After the test result is detected, use glycine or its hydrochloride to elute and repeat the steps for new detection.

[0064] In some embodiments of the present invention, the capture object and the binder include any substance that can specifically bind to the substance to be detected, including: antigen-antibody, enzyme-substrate, complementary pairing sequence and biotin-avidin.

[0065] In some embodiments of the present invention, the capture object and the binding object are antigen-antibody.

[0066] In some embodiments of the present invention, the capture object and the binding object are antibody A and antibody B of the substance to be detected.

[0067] In some embodiments of the present invention, the detection objects of the detection method include proteins, nucleic acid molecules and drug molecules.

[0068] In some embodiments of the present invention, the detection object of the detection method is C-peptide.

[0069] In some embodiments of the present invention, the detection method is an in vitro detection method.

[0070] In some embodiments of the present invention, the detection method does not involve the diagnosis of a disease.

[0071] In some embodiments of the present invention, the detection method is performed based on the above-mentioned detection chip or the above-mentioned detection device.

[0072] The beneficial effects of the present invention are:

[0073] 1. The detection device in the present invention is small and portable, easy to carry, and can effectively realize real-time and rapid detection of any scene, thereby avoiding the defects of high detection costs and inability to perform detection at any time.

[0074] 2. The detection method of the present invention is fast and cyclable, and one round of operation can be completed in about 15 minutes, greatly improving the timeliness and sustainability of the detection; this detection technology does not require professional operation or expensive equipment, and the determination of C-peptide content in the body can be completed using a mobile phone; this detection technology is not limited by time and space and can be detected anytime and anywhere. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Different views of a wearable, portable C-peptide detection device.

[0076] Figure 2 Different views of the microneedle matrix.

[0077] Figure 3 Different views of the microneedle.

[0078] Figure 4 The result of selecting the data readout wavelength is shown in Figure 1, where A is a schematic diagram of selecting the readout wavelength using an ELISA reader, and B is the readable wavelength displayed by the ELISA reader.

[0079] Figure 5 The results of the SPR macromolecular interaction system validation are shown in Figure 2. A is the response value of each concentration of antigen as it is applied, and B is the antibody-antigen regeneration effect for 10 times.

[0080] Figure 6 The actual verification results of the wearable portable C-peptide detection device. A is the image of the OD value change of the C-peptide detection device over time, and BC is the OD value comparison of 10 cycles at 5 minutes.

[0081] Figure 7 SEM images of the microneedle array (A) and a single microneedle tip (B), as well as Masson staining of the microneedle on animal histological sections (C), and microneedle verification test images from 0 min to 10 min (DE).

[0082] Figure 8 is the pressure-displacement curve of the microneedle, where A is the comparison between the pressure and displacement curve of a single microneedle and the force that the stratum corneum can withstand, and B is the magnitude of the force that a single microneedle can withstand when compressed to rupture.

[0083] Fig. 9 The extraction test results of the wearable portable C-peptide detection device, wherein A is the extraction performance of the microneedle-based device within 2 minutes using agarose gel as a skin model (n=8), wherein the inset is a photo of the agarose gel after removing the microneedles, and B is a photo of interstitial fluid extraction using a rabbit model.

[0084] Fig.10The actual product display picture of the wearable portable C-peptide detection device (A) and the schematic diagram of it being equipped on the arm (B).

[0085] Fig.11 Figure 1 shows the training flow chart of the machine learning model used for reading in the mobile phone app (A), the actual operation diagram of the mobile phone app (B), and the actual detection effect diagram (C). DETAILED DESCRIPTION

[0086] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0087] Example 1 A wearable portable C-peptide detection device

[0088] This embodiment provides a wearable portable C-peptide detection device, such as Figure 1 As shown, it mainly consists of three parts, namely a microneedle matrix, a reactor, an injection part and a suction device.

[0089] Wherein, the injection part can be composed of several syringes or injection chambers, which are used to load the reaction solution and transport it to the reactor through the liquid outlet channel. Wherein, according to the type of the loaded liquid, some syringes can be connected in parallel through the liquid outlet channel (of course, they can also be supplied independently). In the present embodiment, a total of 5 syringes are provided as the injection part (syringe 6 is used as a suction device). 5 syringes are connected in parallel through the liquid outlet channel and controlled by a valve (valve 2). By opening valve 2, the corresponding syringe is pushed (and the other syringes are kept stationary) to supply liquid to the reactor through the channel.

[0090] The reactor can be a chamber of any shape. In this embodiment, the reactor is a circular reaction chamber. The reactor is also connected to the external environment through a channel, and a valve (valve 1) is provided on the channel to control whether the channel is connected or not.

[0091] In this embodiment, the suction device is also a syringe, and of course, other suction devices can also be used instead. The main function of the suction device is to provide suction so that the portable C-peptide detection device forms a negative pressure, so that the microneedle matrix can absorb the test sample from the punctured site.

[0092] In this embodiment, the microneedle matrix used has a porous design of microneedles, that is, each microneedle has at least two holes. Figure 2As shown, the microneedle is a double-hole microneedle. The hole runs through the entire microneedle, and the hole is not set at the tip of the microneedle, so that the microneedle can provide effective liquid absorption while maintaining the sharpness to penetrate the skin. The entire microneedle matrix is ​​composed of a number of microneedles, which can be arranged in a regular or irregular manner. In this embodiment, the microneedles are arranged in a matrix in a regular arrangement of 6×6 (such as Figure 3 Of course, commercially available microneedles can also be directly assembled for use.

[0093] The portable C-peptide detection device of this embodiment may further include a fixing device, such as a strap or other device capable of fixing the portable C-peptide detection device to the skin surface, so that it can continuously maintain contact with the skin, so that the microneedle array can obtain the test sample at any time, thereby achieving uninterrupted continuous detection and monitoring.

[0094] The portable C-peptide detection device in this embodiment can be directly obtained by 3D printing or casting. In this embodiment, the microneedle matrix is ​​obtained by 3D printing, and the material can be any conventional commercially available biocompatible resin. The other parts are all obtained by PDMS casting, which can be cast into a mold in one piece or cast into molds separately and then assembled. Figure 1 The schematic diagram is designed and then cast into a mold using PDMS.

[0095] Example 2 Method of using the portable C-peptide detection device

[0096] In this embodiment, a method for using the portable C-peptide detection device in the above embodiment is provided, and the specific steps are as follows:

[0097] (1) Take the cleaned portable C-peptide detection device and load the corresponding reaction solution into each syringe. The reaction solution includes detection antibody, washing solution, color developing solution and eluent. Each reaction solution is loaded into a different syringe (the order of loading, amount and order of syringes are not limited). Since the washing solution needs to be used multiple times during the reaction process, the washing solution can be loaded into multiple syringes or transferred into one syringe.

[0098] In this embodiment, the detection antibody is human C-peptide detection antibody B (purchased from ABCAM), with a concentration of 1 μg / mL, a loading volume of 120 μL, and is loaded in syringe 1. The washing solution is phosphate buffered saline (PBS), which is loaded in syringes 2 and 5, respectively, with a loading volume of 300 μL. The color developing solution is TMB color developing solution, with a loading volume of 300 μL, loaded in syringe 3. The eluent is glycine-hydrochloric acid (also known as glycine hydrochloride or glycine hydrochloride, CAS: 6000-43-7), with a loading volume of 300 μL, loaded in syringe 4.

[0099] (2) Fix the portable C-peptide detection device on the skin. In this embodiment, the selected fixing position is the arm. Of course, other fixing positions can also be selected. When fixing, ensure that the microneedles in the microneedle matrix completely penetrate the skin without any gaps between the microneedle matrix and the skin.

[0100] (3) When using a portable C-peptide detection device to monitor C-peptide concentration, close all valves, pull the piston of syringe 6 to extract the gas in the portable C-peptide detection device, and form a negative pressure, so that the microneedle extracts the test sample (about 20 μL of interstitial fluid) into the reaction chamber. After the extraction is completed, open valve 1 to release the pressure first, and at the same time, open valve 2, push syringe 1, and inject 20 μL of the above-mentioned detection antibody into the reaction chamber. After 10 minutes of reaction, push syringe 2 to inject 50 μL of washing solution into the reaction chamber to terminate the reaction and wash away the unbound antigen and antibody in the mixed solution. Then push the piston of syringe 3 to inject 50 μL of the above-mentioned color developing solution into the reaction chamber. After 5 minutes of reaction, take a photo and read the photo reading (RGB color). After the reading is completed, push the piston of syringe 4 to inject 50 μL of glycine-hydrochloric acid eluent to destroy the bound antigen-antibody connection. After 1 minute of reaction, push syringe 5 to inject 50 μL of washing solution to terminate the elution reaction and wash away the eluted antigens, antibodies, TMB and other substances, providing conditions for the next C-peptide concentration monitoring. When monitoring the C-peptide concentration for the second time, repeat the above operation.

[0101] In the step, all the cleaned materials (with the washing liquid) are discharged from the device (through the channel). Of course, it is also possible to select to independently set a recovery chamber in the device to recycle the cleaned materials.

[0102] The concentration of the measured C-peptide can be obtained by using the standard curve drawn using the standard substance and substituting the reading into it.

[0103] In this embodiment, the specific method for drawing the standard curve is:

[0104] Human C-Peptide ELISA Kit (ab260064, ABCAM, purchased from Guangzhou Xinjin Biotechnology Co., Ltd.) was used to perform Elisa experiments using standard products. The kit was operated according to the instructions for use, and the real-time reading of data was performed in an ELISA reader immediately after the addition of TMB reagent. Two peaks were displayed in the ELISA reader, which were 370nm and 680nm. Using these two wavelengths as reading standards, the OD values ​​of the antigen (standard product) concentrations of 0, 6.25, 12.5, 25, 50, 100, 200, and 400 pg / mL were read respectively, and the concentration standard curve and the change curve of the OD value at each concentration were drawn.

[0105] like Figure 4As shown, it can be found that at a wavelength of 370nm, the OD value peak under the same concentration of antigen is larger and more convenient for daily data reading. Therefore, in actual testing, a wavelength of 370nm is selected as the standard reading wavelength.

[0106] In this embodiment, the absorbance values ​​detected by the portable C-peptide detection device at different C-peptide concentrations can clearly distinguish the concentrations of the standard in 5 minutes. Combined with the time used in the method of use, the test results can be obtained in only 15 minutes.

[0107] Example 3: Effect verification of portable C-peptide detection device

[0108] (1) Recyclability of portable C-peptide detection device:

[0109] The recyclability of the portable C-peptide detection device in the above example was verified using a SPR macromolecular interaction system (Biacore 8k (Cytiva)).

[0110] Using standard samples as antigens, the above antigen-antibody binding and glycine hydrochloride eluent elution were repeated at different concentrations in the SPR macromolecular interaction system for cyclability testing. The test was repeated multiple times to test whether the repeated binding and dissociation between the antigen and the antibody fixed in the reaction chamber would affect the detection effect.

[0111] The results are as follows Figure 5 shown.

[0112] It can be found that based on Figure 5 The response values ​​of the antigens at different concentrations over time reflect the binding and dissociation process between the antigen and the antibody fixed on the chip surface. The force between the antibody and the antigen is at the level of nanomolar. After 10 repeated binding and elution, it can be found that complete elution can be achieved each time, and the elution rate is 100%.

[0113] Subsequently, the inventor used the portable C-peptide detection device to perform another verification, repeating the above binding and elution process. The standard concentration of 100 pg / mL was used as the antigen to be tested. After 10 repeated binding and elution with the eluent, the results were as follows: Figure 6 shown.

[0114] It can be found that after 10 repeated combinations and eluent elutions, the changes between the detection signals detected each time are quite stable. When the time point of 5min is used as a benchmark, the OD value at this time point is maintained at about 0.3, which is relatively stable. Therefore, by SPR macromolecular interaction experiments and actual use verification, it can be explained that the use of the glycine eluent in the embodiment of the present invention can completely elute the antigen already connected to the antibody, thereby re-exposing the antibody in the reaction chamber, achieving a recyclable effect, and the number of repeated elutions is not less than 10 cycles.

[0115] (2) Sample extraction capability of portable C-peptide detection device:

[0116] A scanning electron microscope (SEM) was used to obtain SEM images of the microneedle matrix and the tip of a single microneedle of the portable C-peptide detection device in the above example.

[0117] like Figure 7 As shown, the microneedles of the portable C-peptide detection device have a conical structure that allows safe and effective penetration. The needle spacing between adjacent tips, that is, the distance from center (needle tip) to center (needle tip), is finally 1200μm after structural optimization. The spacing is not too close, resulting in too high a density of needles to effectively insert into the skin. The conical microneedle tip has two hollow channels with a diameter of 150μm. The bottom diameter of the microneedle is 600μm, and the vertical height from the tip to the bottom is 650μm.

[0118] After histological examination of the skin tissue after microneedle puncture using a portable C-peptide detection device, it was found that the tip of the microneedle can pierce the epidermis to reach the dermis, but it will not enter the capillaries and nerve endings, so the dermal ISF (interstitial fluid) can be extracted painlessly. In addition, through histological examination, it was found that after being punctured by the microneedle, due to the reasonable size design of the microneedle, the skin will show the ability to reseal within 10 minutes after the microneedle is removed, indicating that the degree of damage to the skin is low ( Figure 7 ).

[0119] The microneedles were further pressure tested to measure their positions under different pressure conditions and to draw pressure and displacement curves.

[0120] The results are as follows Figure 8 It can be found that the force that each microneedle in the microneedle matrix in the embodiment of the present invention can withstand is about 30N, which is far more than the 0.058N that the stratum corneum can withstand. Therefore, it can be determined that the microneedles in the embodiment of the present invention can fully meet the requirements for extracting interstitial fluid by pressing.

[0121] Finally, the inventors used 1.5wt.% agarose gel as a skin model to verify the extraction amount, and used rabbits as living experimental subjects for further verification.

[0122] The results are as follows Fig. 9 shown.

[0123] It can be found that by only using the pressing method for extraction, 75 μL of simulated interstitial fluid can be obtained in the 1.5wt.% agarose gel skin model. When rabbits are used as living experimental subjects, the amount of interstitial fluid extracted can also reach at least 50 μL, which is enough to meet the amount of liquid used for analysis by the device.

[0124] (3) Actual detection effect of portable C-peptide detection device

[0125] The portable C-peptide detection device prepared in the above embodiment is used in actual detection. The actual product and the representative display diagram of the sampling detection on the arm of the subject in this embodiment are shown in the figure below. Fig.10 shown.

[0126] The detection method and product design are the same as those in the above embodiment. The specific machine learning model training process is as follows: Fig.11 In this embodiment, a mobile phone is used as a terminal to directly calculate the C-peptide content by directly reading the color feature (RGB), as shown in the schematic diagram Fig.11 As shown in B.

[0127] Test results such as Fig.11 C shows the R of the linear regression model trained by machine learning. 2 =0.97, which has good prediction performance, indicating that the product in the above embodiment has good detection effect.

[0128] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A detection chip, characterized in that: The detection chip comprises a microneedle array, a reaction chamber and an injection part which are sequentially connected through a liquid outlet channel; The microneedle array is used to obtain a test sample from the skin; The reaction chamber is pre-buried with a fixed capture object, and the capture object is used to capture the substance to be detected in the detection sample; The injection part is loaded with a reaction liquid, and is used to store and drive the reaction liquid stored therein to react with the substance to be detected captured by the capture object in the reaction chamber; Wherein, the reaction solution comprises a conjugate, a developer, a washing solution and an eluent; The binding substance is used to bind to the substance to be detected or the substance to be detected-capture complex; The eluent is used to dissociate the substance to be detected and the captured object; The eluent is glycine or its hydrochloride.

2. The detection chip according to claim 1, characterized in that: The microneedles in the microneedle array are porous microneedles with 2 to 3 holes, a hole diameter of 120-180 μm, a microneedle spacing of 1100-1300 μm, a microneedle bottom diameter of 500 to 700 μm, and a vertical height of 500-700 μm.

3. The detection chip according to claim 1, characterized in that: The capture object and the binder include any substance that can specifically bind to the substance to be detected, including: antigen-antibody, enzyme-substrate, complementary pairing sequence and biotin-avidin; preferably antigen-antibody.

4. The detection chip according to claim 1, characterized in that: The detection chip also includes a suction device and a pressure release channel; the suction device is used to assist the microneedle array in obtaining the detection sample, and the pressure release channel is used to release the pressure on the detection chip; Preferably, a controllable switch is provided on the pressure release channel, and the controllable switch comprises a valve device.

5. The detection chip according to claim 1, characterized in that: The liquid outlet channel is also provided with a controllable switch for controlling the flow of the fluid in the injection part to the reaction chamber; preferably, the controllable switch comprises a valve device.

6. A detection device, characterized in that: The detection device comprises the detection chip according to any one of claims 1 to 5 and a fixing device, wherein the fixing device is used to fix the detection chip on the surface of the part to be sampled.

7. Use of the detection chip according to any one of claims 1 to 5 or the detection device according to claim 6 in in vitro real-time detection or monitoring; the in vitro real-time detection or monitoring does not involve diagnosis of a disease.

8. The use according to claim 7, characterized in that: The real-time detection or monitoring is the real-time detection or monitoring of C-peptide.

9. A recyclable detection method comprising: Add the test sample to the pre-embedded capture object, add the conjugate, wash to remove the unbound test sample and conjugate, elute with glycine or its hydrochloride after the test result, and repeat the steps again for new test; Wherein, the detection method does not involve the diagnosis of the disease.

10. The detection method according to claim 9, characterized in that: The detection method is performed based on the detection chip described in any one of claims 1 to 5 or the detection device described in claim 6.

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